2.1. Recombinant DNA Technology
The development of recombinant DNA procedures, which are often referred to as gene-splicing or genetic engineering, has made possible the production of a wide variety of biological products. In current recombinant DNA procedures, specific DNA sequences are inserted into an appropriate DNA vehicle, or vector, to form recombinant DNA molecules that can replicate in host cells. Circular double-stranded DNA molecules called plasmids are frequently used as vectors, and the preparation of such recombinant DNA forms entails the use of restriction endonuclease enzymes that can cleave DNA at specific base sequence sites.
Once cuts have been made by a restriction enzyme in a plasmid and in a segment of foreign DNA that is to be inserted, the two DNA molecules may be covalently linked by an enzyme known as a ligase. General methods for the preparation of such recombinant DNA molecules have been described by Cohen et al. [U.S. Pat. No. 4,237,224], Collins et al. [U.S. Pat. No. 4,304,863] and Maniatis et al. [Molecular Cloning: A Laboratory Manual, 1982, Cold Spring Harbor Laboratory]. Because they illustrate much of the state of the art, these references are hereby incorporated by reference.
Recombinant DNA molecules can be used to produce the product coded for by the inserted gene sequence only if a number of conditions are met. Foremost is the requirement that the recombinant molecule be compatible with, and thus capable of autonomous replication in, the host cell. Much recent work has utilized the bacterium Escherichia coli (E. coli) as a host organism because it is compatible with a wide range of recombinant plasmids. Depending upon the vector/host cell system used, the recombinant DNA molecule is introduced into the host by transformation, transduction or transfection.
The mere insertion of a recombinant vector into a host cell will not in itself assure that significant amounts of the desired gene product will be produced. For this to occur, the foreign gene sequence must be fused in proper relationship to a signal region in the vector for DNA transcription called a promoter Alternatively, the foreign DNA may carry with it its own promoter, as long as it is recognized by the host. Whatever its origin, the promoter is a DNA sequence that is "upstream" of the foreign gene that is to be expressed which directs the binding of RNA polymerase and therefore "promotes" the transcription of DNA to messenger RNA (mRNA).
Given strong promotion that can provide large quantities of mRNA, the ultimate production of the desired gene product will depend upon the effectiveness of translation from mRNA to protein. This, in turn, is dependent upon the efficiency of ribosomal binding to the mRNA and upon the stability of the mRNA within the host cell. In eukaryotic cells, the factors governing translational efficiency are poorly understood but appear to include a favorable nucleic acid sequence surrounding an AUG codon which initiates translation [Kozak, Cell 44:283 (1986)]. Factors affecting the stability of the mRNA, which are poorly understood in both prokaryotic and eukaryotic cells, are also critical to the amount of protein production that can be obtained.
Most of the work in the recombinant DNA field to the present has focused on the use of bacterial expression systems such as E. coli. Yet, the use of bacterial cells has a number of undesirable aspects. For example most proteins and polypeptides produced in E. coli. accumulate in the periplasmic space. Recovery of these gene products thus requires disruption of the cells, a process which is inefficient and which leads to a serious purification problem, as the desired product must be purified from the numerous other E. colicellular constituents. Also, bacteria cannot carry out glycosylation which is needed to complete the synthesis of many interesting gene products or form the specific disulfide bonds which are essential for the proper conformation and biological activity of many eukaryotic proteins.
To overcome these deficiencies in bacterial expression systems, the attention of genetic engineers is increasingly turning to the use of eukaryotic host cells for recombinant DNA. Cells such as yeast and mammalian cells can secrete desired gene products into the culture medium and can carry out essential glycosylation processes as well. Yet, the use of mammalian cells for recombinant DNA cloning and expression also poses a host of technical obstacles that must be overcome. For example, the endogenous plasmids that have proven to be so useful in bacteria are not replicated by higher eukaryotic cells. As a result, other approaches must be taken.
One approach has been to use the lower eukaryotic yeast. Saccharomyces cerevisiae, which can be grown and manipulated with the same ease as E. coli. Yeast cloning systems are available, and through the use of such systems the efficient expression in yeast of a human interferon gene has been achieved [Hitzeman et al., Nature (London) 293:717 (1981)]. Interferon genes do not contain introns, however, and it has been found that yeast cells do not correctly transcribe at least one heterologous mammalian gene that does contain introns, the rabbit .beta.-globin gene (Beggs et al., Nature (London) 283:835 (1980)].
In another approach, foreign genes have been inserted into mammalian cells by means of direct uptake. This has been accomplished, for example, by calcium phosphate co-precipitation of cloned genes, by which procedure about 1-2% of the cells can generally be induced to take up the DNA. Such a low level of uptake, however, produces only a very low level of expression of the desired gene product. Where mammalian cells can be found which lack the thymidine kinase gene (tk.sup.- cells), better results can be obtained by co-transformation. Tk.sup.- cells, which cannot grow in selective HAT (hypoxanthine-aminopterin-thymidine) medium, can regain this lost enzymatic activity by taking up exogenous DNA (such as herpes simplex viral DNA) containing the tk gene through calcium phosphate co-precipitation. Other DNA covalently ligated to the tk DNA or merely mixed with it will also be taken up by the cells and will often be co-expressed [see Scangos et al., Gene 14:1 (1981)].
In a third approach, viral genomes have been used as vectors for the introduction of other genes into mammalian cells, and systems based upon Simian virus 40, papillomavirus.. and adenovirus genomes have been described [see P.W.J. Rigby, Expression of Cloned Genes in Eukaryotic Cells Using Vector Systems Derived from Viral Replicans, in Genetic Engineering, Vol. 3, R. Williamson, ed., Academic Press, New York, pp. 83-141 (1982) for a review]. These systems, however, suffer from the drawback of limited host cell range. Moreover, viral replication in these systems leads to host cell death. The use of retroviral DNA control elements avoids many of the disadvantages of these viral vector systems
Gorman et al. [Proc. Natl. Acad. Sci. U.S.A. 79:6777 (1982)) have shown, for example, that the Rous sarcoma virus long terminal repeat (LTR) is a strong promoter that can be introduced into a variety of cells, including CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells and mouse NIH/3T3 cells by DNA-mediated transfection.
Evidence for the regulation of gene expression by 5' and 3' noncoding sequences has come from the study of oncogenes and the mRNAs of higher eukaryotic cells in which the results of the elimination or modification of these sequences on gene expression have been observed. For example, Treisman [Cell 42:889 (1985)] has studied the accumulation of c-fos RNA following serum stimulation of mouse fibroblasts into which a cloned human c-fos gene (the cellular homolog of the oncogene carried by the FBJ murine osteosarcoma virus, designated c-fos.sup.H) had been transfected. Ordinarily, serum stimulation of such cells causes a strong but transient burst of c-fos mRNA which reaches a maximum by 10 to 15 minutes and decreases rapidly thereafter, reaching prestimulation levels within 1 to 2 hours due to rapid degradation of the mRNA.
When the c-fos.sup.H 5' flanking sequences are fused to heterologous genes in the absence of the normal c-fos.sup.H 3' flanking sequences, however, the resulting genes are still inducible by serum factors but the mRNA thereby produced persists for up to 4 hours following serum stimulation. Experiments which hybrid transcription units show that only genes containing the 3' end of the c-fos.sup.H gene and the 3' noncoding regions show the typical rapid decrease in mRNA levels following stimulation. It may thus be that the c-fos 3' sequences act to destabilize fusion gene RNA. and their elimination or modification may have a positive effect upon gene expression.
Further evidence for a regulatory role by 3' noncoding sequences has come from studies by Simcox et al. [Mol. Cell. Biol. 5:3397 (1985) on the Drosophila melanogaster heat shock protein system. When shifted from growth at ambient temperature to 37.degree. C., Drosophila melanogaster rapidly produces a number of "heat shock" proteins, among which is a major protein called hsp 70, through a process in which new mRNAs are produced and rapidly transcribed. Following a return to normal growth temperatures, transcription of the hsp 70 gene is rapidly repressed and the levels of the corresponding mRNA rapidly decline, thereby quickly terminating further hsp 70 protein production. Simcox et al. found, however, that the rapid repression of hsp 70 protein synthesis after release from heat shock is delayed when 3' sequences have been deleted, suggesting that the 3' sequences normally act to destabilize the hsp 70 mRNA after the temperature downshift.
Evidence pointing to an mRNA regulatory role has also been obtained for 5' noncoding sequences. Butnick et al. [Mol. Cell. Biol. 5:3009 (1985)] have shown that a 5' noncoding sequence containing about 550 bases (designated exon 1) of the human c-myc gene (the cellular homolog of the avian myelocytomatosis virus oncogene) affects the expression of plasmids bearing that gene in CV1 monkey kidney cells transformed with an origin-defective Simian virus 40 (designated COS cells). Transcripts from plasmids in which the 5' noncoding sequences of the c-myc had been deleted were found to be present at a higher steady-state level than were transcripts from plasmids bearing the intact gene, suggesting that the 5' noncoding sequences in some way act to destabilize the corresponding mRNA.
In another study, Rabbitts et al. [EMBO J. 4:3727 (1985)] have shown that the truncation of exon 1 from the c-myc gene causes an increase in the stability of c-myc mRNA in COLO 320 cells. Similarly, Eick et al. [EMBO J. 4:3717 (1985)] have demonstrated that mRNAs produced in Burkitt's lymphoma cells by c-myc genes in which there has been a translocation in exon 1 are much more stable than the normal mRNAs.
The above references all suggest that 5' and 3' noncoding regions of a variety of genes may in some way produce instability in the corresponding mRNAs transcribed from these genes. Deletion or alteration of these noncoding regions in these cases produced increased mRNA stability and, hence, an increased overall level of gene expression. Yet the effect of modification or deletion of such noncoding sequences on gene expression cannot be predicted with assurance.
For example, Johansen et al. [Proc. Natl. Acad. Sci. U.S.A. 81:7698 (1984) have varied the length of the 5' noncoding leader region in a recombinant vector system containing gene control elements fused to the Escherichia coli galactokinase (galk) gene. The variation in length of the noncoding region had no effect on galk expression. Similarly, Katz et al. [Mol. Cell. Biol. 6:372 (1986)] have introduced both deletions and substitutions of other sequences into the 5' untranslated leader of avian retroviral mRNAs. Generally, these deletions and substitutions caused a substantial decrease in the expression of the env gene. These decreases in expression, however, were not due to reductions in mRNA levels. It appears instead that the changes in the noncoding segments caused a translational deficiency which led to the overall reduction in expression.